When glucose and protein react in the blood, stable fructosamines are formed through the unstable Shiff bases. The concentration of fructosamines in the blood has correlation with the concentration of glucose in the blood. Further, it is hardly affected by even short-term variations in the glucose concentration. Therefore, it is known to be extremely effective to measure the concentration of fructosamines in the blood so as to diagnose diabetes and monitor the progress of treatment of diabetics.
Japanese Examined Patent Publication (Kokoku) No. 1-13062 describes a method for measuring the concentration of fructosamines in a blood sample by using the fact that fructosamines have reducing activity under alkaline conditions and cause a color agent (for example, a tetrazolium salt) to produce color. This method is preferable in the point that it can be applied to an automatic measurement apparatus. However, blood samples contain reducing substances (for example, ascorbic acid, glutathione, and uric acid) in addition to fructosamines. Therefore, it was necessary to perform the measurement after a certain time elapses from the start of the reaction so as to reduce or avoid the effects of these nonspecific reducing substances.
Various proposals were made on techniques for eliminating the influence of coexisting substances. For example, Japanese Unexamined Patent Publication No. 63-15168 describes a method for removal of the nonspecific reducing components and turbidity inducing components by making use of a neutral pH value. Further, Japanese Unexamined Patent Publication No. 63-304999 describes a method for removing the nonspecific reducing components and turbidity inducing components by uricase or a surface active agent and performing the measurement in a single stage. Further, Japanese Unexamined Patent Publication No. 63-182567 describes a method for measuring fructosamines after performing a step for removing interfering substances in advance and Japanese Unexamined Patent Publication No. 1-108997 describes a method for removing interfering substances by an agent oxidizing enzymes, etc.
However, in the methods described in the above patent publications, a tetrazolium salt is used as the optimal coloring agent in the same way as the method described in said Japanese Unexamined Patent Publication No. 1-13062. This tetrazolium salt produces the water insoluble coloring substance, formazane, as a result of a reaction. Formazane easily adheres to equipment of a measuring apparatus and is difficult to remove. Further, the surface active substances coexisting in a liquid sample (for example, lipids and proteins) result in a change of the apparent absorbance of formazane, so the measurement results are affected by lipids and proteins and there was the problem of a reduction in the measurement precision.
The present inventors engaged in studies to resolve the above problems and as a result discovered that if a specific compound is brought into contact with fructosamines, active oxygen is produced and hydrogen peroxide is derived from that active oxygen, so by measuring the amount of the hydrogen peroxide, it is possible to measure the fructosamines. The present invention is based on this discovery.